Lack of Benefit of Early Protocol Biopsies in Renal Transplant Patients Receiving TAC and MMF: A Randomized Study

Authors


* Corresponding author: David N. Rush, drush@exchange.hsc.mb.ca

Abstract

We conducted a randomized, multicenter study to determine whether treatment of subclinical rejection with increased corticosteroids resulted in beneficial outcomes in renal transplant patients receiving tacrolimus (TAC), mycophenolate mofetil (MMF) and prednisone. One hundred and twenty-one patients were randomized to biopsies at 0,1,2,3 and 6 months (Biopsy arm), and 119 to biopsies at 0 and 6 months only (Control arm). The primary endpoint of the study was the prevalence of the sum of the interstitial and tubular scores (ci + ct)> 2 (Banff) at 6 months. Secondary endpoints included clinical and subclinical rejection and renal function. At 6 months, 34.8% of the Biopsy and 20.5% of the Control arm patients had a ci + ct score ≥ 2 (p = 0.07). Between months 0 and 6, clinical rejection episodes were 12 in 10 Biopsy arm patients and 8 in 8 Control arm patients (p = 0.44). Overall prevalence of subclinical rejection in the Biopsy arm was 4.6%. Creatinine clearance at 6 months was 72.9 ± 21.7 in the Biopsy and 68.90 mL/min ± 18.35 mL/min in the Control arm patients (p = 0.18). In conclusion, we found no benefit to the procurement of early protocol biopsies in renal transplant patients receiving TAC, MMF and prednisone, at least in the short term. This is likely due to their low prevalence of subclinical rejection.

Introduction

Banff histological criteria for acute rejection (1) are present in a variable proportion of patients with normal graft function in whom protocol biopsies are obtained. A single center randomized study showed that treatment of subclinical rejection with corticosteroids was associated with improved renal transplant outcomes in patients receiving cyclosporine (CsA), azathioprine and prednisone. Specifically, patients randomized to early (months 1–3) protocol biopsies had a significant decrease in clinical acute rejection episodes, a reduced chronic tubulo-interstitial score at 6 months and a lower serum creatinine at 24 months than patients in whom protocol biopsies were not performed. The prevalence of subclinical rejection in months 1–3 in these patients was approximately 30% (2).

A subsequent study using the microemulsion (ME) formulation of CsA in combination with MMF and prednisone showed a decrease in clinical but not subclinical rejections, compared to patients treated with CsA, azathioprine and prednisone (3). Similarly, a study that utilized TAC, azathioprine and prednisone reported a decrease in clinical rejection episodes as compared to CsA-ME, azathioprine and prednisone, but no effect on the prevalence of subclinical rejection (4).

In the present randomized, multicenter study we sought to determine the prevalence of subclinical rejection in protocol biopsies performed in months 1–3 in renal transplant patients receiving TAC, MMF and prednisone as baseline immunosuppression, and tested the hypothesis that its treatment would have a beneficial effect on clinical and histological outcomes.

Materials and Methods

Patients

Patients were recruited from 11 Canadian centers in the provinces of British Columbia (2 sites), Alberta (2 sites), Saskatchewan (1 site), Manitoba (1 site), Ontario (2 sites) and Quebec (3 sites). The one site in the United States was in California. Recruitment to the study began in September 2001 and extended until February 2004. Two hundred and twenty-nine patients were recruited in Canada and 11 in California.

Inclusion criteria were: recipients of first or second HLA mismatched (≥1 mismatch) renal transplant from a deceased or living donor; age over 18 years; negative pregnancy test and pregnancy avoidance. Exclusion criteria were: recipients of a kidney from a donor over 65 years of age; peak pretransplant% panel reactive antibody (PRA) > 50; previous graft loss to rejection within a year from transplant; use of an investigational drug within 3 months of the study; pregnancy or breastfeeding; hypersensitivity to any drugs in the immunosuppressive protocol, related drugs or their excipients; patients receiving a kidney lacking a pre-implantation biopsy; and patients with significant disease or disability that might prevent adherence to the protocol.

Study design

The study was an open-label, randomized, multicenter parallel group study that compared two biopsy regimens. Patients were randomized under concealed allocation to protocol biopsies at 1, 2, 3 and 6 months (Biopsy arm) or to biopsies at 6 months only (Control arm). All patients had biopsies at implantation. Randomization was in a ratio of 1:1 to the two arms, and was stratified using the minimization method for donor source (living or deceased), donor age (<35, 35–55, >55), and center. The study was approved by the IRB at each institution. Patients will be followed up to 24 months when another protocol biopsy and renal function data will be obtained.

Immunosuppression in both patient arms was a standardized triple therapy regimen. This consisted of TAC at an initial oral dose of 0.15 to 0.30 mg/kg/day bid, MMF, starting at 1g bid, and prednisone. Target trough levels for TAC were 12 ng/mL ± 2 ng/mL between weeks 1 and 2, 10 ng/mL ± 2 ng/mL between week 3 and month 3 and 8 ng/mL ± 2 ng/mL for months 4–6. The dose of MMF was adjusted as necessary. Prednisone was started at 1 mg/kg, tapering to 20 mg by month 1, 10 mg by month 2, 7.5 mg by month 3 and 5 mg by months 4–6.

Rejection episodes defined either on clinical grounds or by pathological criteria, were treated with a 2-week tapering course of oral prednisone starting at 200 mg. Subclinical rejection, defined as a Banff score ≥ (ai)2 (at2) (acute interstitial, acute tubulitis) in a protocol biopsy obtained in a patient with <10% change in serum creatinine from baseline, was treated similarly. Methylprednisolone and anti-lymphocyte agents were used at the discretion of the investigators.

Renal biopsies were obtained with an 18 gauge spring-loaded gun under ultrasound guidance. Two cores of tissue were obtained and were scored using the 1997 Banff schema (1). The local pathologist was blinded to the type of biopsy (protocol or ‘for cause’). Treatment decisions were based on these local readings; however, implantation and 6-month biopsies were sent for blinded central review to the University of Calgary.

Efficacy and safety considerations

The primary endpoint of the study was the prevalence of chronic histology, defined as the sum of the interstitial and tubular scores (ci + ct) of ≥ 2 at 6 months. Secondary endpoints were the prevalence of subclinical rejection at 6 months, the frequency of biopsy-confirmed or suspected acute rejections during the periods 0–6 months and renal function at 6 months posttransplant, as determined by serum creatinine and by 24-h urine creatinine clearance (calculated using the Cockroft–Gault formula) and protein excretion.

Patient and graft survival, adverse events, including biopsy complications, and the use of anti-hypertensive, lipid-lowering and hypoglycemic agents were monitored throughout the study by a Data Safety Monitoring Board.

Statistical analysis

Sample size was based on the anticipated prevalence of the chronic score of ci + ct ≥ 2 at 6 months of 5% in the Biopsy arm and 20% in the Control arm, observed in a previous study (2). With these assumptions, at least 88 evaluable patients in each arm would be required to detect differences with a power of 0.80 and a type-I error of 0.05. To compensate for dropouts from the efficacy analysis, 100 patients were randomized into each arm of the study; however, an additional 20 in each arm had to be included because of a greater than expected noncompliance with the biopsy protocol.

All data were stored as SAS datasets. All analyses (including statistical tables and listings) were done using 'SAS Institute, Cary, NC' v9.1 software.

Results are presented as means ± SD for continuous variables. Frequencies of categorical variables are given as counts and percentages. For continuous variables, the two sample t-test for normal data or Wilcoxon rank-sum test for nonnormal data was used. For categorical variables, the chi-square and Cochran-Mantel-Haenszel test were used.

Logistic regression was performed to assess the independent effects of correlates of ci + ct at 6 months (dichotomized to <2 or ≥2) as the response variable. The explanatory variables were: treatment arm (Biopsy vs. Control); site; donor age (≤ 50 vs. >50—the median value); donor status (living vs. deceased); donor gender (female vs. male); patient age (≤ 50 vs. >50); patient gender (female vs. male); HLA mismatches (≥5 vs. <5); %PRA (0 vs. >0 vs. unknown); cold ischemia time (≤10:41 h vs. >10:41 h—the median value); delayed graft function (yes vs. no); donor ci + ct score at implantation (>0 vs. 0); donor ct score at implantation (>0 vs. 0); donor death due to subarachnoid hemorrhage (yes vs. no vs. other/unknown); donor hypertension (present vs. absent vs. unknown) and donor's terminal creatinine (≤66 μmol/L vs. >66 μmol/L—the median value—vs. unknown). The variables with p-value less than 0.05 obtained in the full model were used in the reduced model using the stepwise elimination method. Treatment arm, site, donor age and donor status were included in the reduced model as they were stratification variables used in the randomization.

Results

Patient demographics and disposition

Of the 240 patients randomized, all 22 from one center (10 randomized to the Biopsy arm and 12 to the Control arm) were excluded because of the use of an induction agent not allowed for in the immunosuppressive protocol. The demographics of the remaining (intent to treat; ITT) patients are shown in Table 1. There were 36 early discontinuations from the study between baseline and month 6, 27 in the Biopsy arm and 9 in the Control arm. Most discontinuations were withdrawals of consent (15 patients; 13 in the Biopsy arm), followed by protocol violations (5 patients), adverse events (4 patients) and graft loss (3 patients). This left a total of 84 patients in the Biopsy arm and 98 in the Control arm that completed the 6-month study visit and are the subject of this report. These data are shown in Table 2.

Table 1.  Subject demographics
ParameterBiopsy arm (n = 111)Control arm (n = 107)
Age [years] (mean ± SD)47.7 (11.8)  47.7 (13.6)  
Male/Female (N)74/3776/31
Race: N (%)
 Aboriginal6 (5.4)4 (3.7)
 Asian10 (9.0) 17 (15.9)
 Black1 (0.9)2 (1.9)
 Caucasian88 (79.3)79 (73.8)
 Hispanic2 (1.8)0 (0.0)
 Other4 (3.6)5 (4.7)
Primary reason for transplant
 Glomerulosclerosis2 (1.8)9 (8.4)
  Interstitial disease5 (4.5)2 (1.9)
  Systemic autoimmune disease4 (3.6)3 (2.8)
  Diabetic nephropathy22 (19.8)22 (20.6)
  Hereditary kidney disease7 (6.3)10 (9.3) 
  Glomerulonephritis18 (16.2)18 (16.8)
  Hypertension3 (2.7)4 (3.7)
 Drug toxicity1 (0.9)0 (0.0)
  Congenital disorder2 (1.8)0 (0.0)
 Obstruction1 (0.9)1 (0.9)
  Other46 (41.4)38 (35.5)
Cold ischemia Time [min] (mean ± SD)666.6 (418.8)  626.6 (410.7)  
PRA level [%] (mean ± SD)2.5 (9.0) 1.0 (2.5)  
Table 2.  Subject disposition
 No. (%) of subjects
Biopsy armControl armAll
Total number of subjects111107218
Number of early discontinuations27 (24.3)9 (8.4)36 (16.5)
Reasons for discontinuation:
 Patient withdrew consent13 (11.7)2 (1.9)15 (6.9) 
 Violation of enrollment criteria1 (0.9)0 (0.0)1 (0.5)
 Protocol violation3 (2.7)2 (1.9)5 (2.3)
 Lost to follow-up0 (0.0)0 (0.0)0 (0.0)
 Adverse event3 (2.7)1 (0.9)4 (1.8)
 Graft loss1 (0.9)2 (1.9)3 (1.4)
 Death1 (0.9)0 (0.0)1 (0.5)
 Other6 (5.4)3 (2.8)9 (4.1)

Three patient populations were analyzed. The ITT population of 218 patients, consisted of 111 in the Biopsy arm and 107 in the Control after exclusion of the 22 patients that had an early protocol violation. The ‘ITT = biopsied’ population of 192 patients, consisted of 90 patients in the Biopsy arm and 102 patients in the Control arm that remained in the study after 1 month and had at least 1 protocol biopsy performed (at month 1, 2 or 3) if in the Biopsy arm. Finally, a third population of 151 patients consisted of 72 patients in the Biopsy arm and 79 patients in the Control arm that had both a baseline and 6-month protocol biopsy. The demographics of these latter 2 patient groups did not differ from those of the ITT group (not shown).

TAC, MMF and prednisone doses were not different at any time-point between the Biopsy and Control arm patients (not shown). TAC trough levels at all time points were similar between the patient groups (Figure 1). Concomitant medications, such as anti-virals, anti-hypertensives (including drugs affecting the renin-angiotensin system), lipid-lowering and hypoglycemic agents did not differ between the Biopsy and Control arm patients (not shown).

Figure 1.

FK506 trough levels.

Patient and graft survival

There were two graft losses (including one due to patient death) in the Biopsy arm, and two graft losses and no patient deaths in the Control arm, for a greater than 98% patient and graft survival in the ITT population. All graft losses occurred in the first month of the study. Three were due to vascular causes (infarction, graft necrosis and renal vein thrombosis), and one was due to patient death from ventricular fibrillation.

Delayed graft function

Delayed graft function (DGF) (defined as the need for dialysis in the first week posttransplant) occurred in 17 patients in the Biopsy arm (15.3%) and in 18 patients in the Control arm (16.8%) (p = 0.76 chi-square test; ITT population).

Acute rejection episodes

Clinical rejections occurred in the first month posttransplant, in 8 patients in the Biopsy arm (9%) and in 7 patients in the Control arm (6.9%), respectively (p = 0.61). Between months 1 and 6 there were four additional rejections in three Biopsy arm patients and only one in one Control arm patients. The overall acute rejection rate (ITT population) was therefore 12 episodes in 10 Biopsy arm patients and 8 episodes in 8 Control arm patients (p = 0.44). All rejections were biopsy-proven.

Histological outcomes

Compliance with the biopsy protocol.  The compliance with baseline and protocol biopsies for the ITT population is shown in Table 3, and that of the patients that had both baseline and 6-month biopsies is shown in Table 4. The biopsy samples were ‘adequate’ or ‘marginal’ by Banff criteria in approximately 83% of cases at baseline and in approximately 87% at 6 months (ITT population).

Table 3.  Compliance with protocol biopsies (ITT)
Study periodNo. (%) of subjects
Biopsy arm (n = 111)Control arm (n = 107)
Subjects availableBiopsies performedSubjects availableBiopsies performed
Implant111 105 (94.6) 107 101 (94.4) 
Month 19870 (71.4)
Month 29567 (70.5)
Month 39274 (80.4)
Month 68479 (94.0)9883 (84.7)
Table 4.  Compliance in patients that had both baseline and 6 months biopsies
Study periodNo. (%) of subjects
Biopsy arm (n = 72)Control arm (n = 79)
Subjects availableBiopsies performedSubjects availableBiopsies performed
Implant72 72 (100.0)7979 (100.0)
Month 17258 (80.6)
Month 27262 (86.1)
Month 37266 (91.7)
Month 672 72 (100.0)7979 (100.0)

Implantation biopsies.  Implantation biopsies of the ITT-biopsied population showed essentially no acute inflammatory changes in either the Biopsy or Control arm patients. Eighty-seven percent of the Biopsy arm patients and 74% of the Control arm patients, respectively, had chronic scores of zero; and 97% of biopsies in both arms showed ci + ct scores of <2. The sum of the chronic score (ci + ct) at baseline was 0.18 ± 0.62 in the Biopsy (n = 69) and 0.25 ± 0.52 in the Control arm patients (n = 82), respectively (p = 0.69).

Subclinical rejection.  Subclinical rejection was infrequent, occurring in 4.6% of the biopsies overall in the Biopsy arm patients, with an increasing prevalence in the latter months. The prevalence of subclinical rejection in the 6-month protocol biopsy was 9% in the Biopsy arm and 6% in the Control arm, respectively (p = 0.48). Borderline rejection was infrequent. These results are shown in Table 5. Between the baseline and the 6-month biopsies (ITT-biopsied population), the ai score increased significantly in both the Biopsy and Control arms by a mean

Table 5.  Subclinical rejection (ITT biopsied)
Study period No. (%) of subjects
Biopsy arm (n = 90)Control arm (n = 102)
ImplantSubclinical acute/active rejection0 (0.0)0 (0.0)
Subclinical borderline rejection with treatment0 (0.0)0 (0.0)
Month 1Subclinical acute/active rejection4 (5.7)
Subclinical borderline rejection with treatment1 (1.4)
Month 2Subclinical acute/active rejection0 (0.0)
Subclinical borderline rejection with treatment2 (3.0)
Month 3Subclinical acute/active rejection6 (8.1)
Subclinical borderline rejection with treatment1 (1.4)
Month 6Subclinical acute/active rejection7 (8.9)5 (6.0)
Subclinical borderline rejection with treatment0 (0.0)0 (0.0)

0.51 ± 0.74 and 0.38 ± 0.68, and the at score increased by a mean 0.56 ± 0.91 and 0.35 ± 0.76, in the Biopsy and Control arms, respectively (p < 0.0001 for both groups vs. baseline; p = 0.15 between groups for ai and at score). These comparisons were similar in the group that had biopsies both at baseline and at 6 months (data not shown).

Chronic histology.  The mean ci + ct score at 6 months was 1.17 ± 1.32 in the Biopsy arm and 0.79 ± 1.11 in the Control arm (p = 0.09). The proportion of patients with ci + ct scores ≥ 2 was 34.8% in the Biopsy arm and 20.5% in the Control arm (p = 0.07). Between the baseline and 6-month biopsies (ITT-biopsied group), the mean ci + ct score increased significantly by 1.12 ± 1.36 and 0.57 ± 1.02, in the Biopsy and Control arms, respectively (p < 0.0001 for both groups vs. baseline; p = 0.04 between the groups). For those patients that had both an implantation biopsy and a 6-month biopsy the change in mean ci + ct score was significant, favoring the Control arm (p < 0.04). Notably however, six patients in the Control arm and one patient in the Biopsy arm improved their chronic score between the implantation and 6 month biopsies.

The multivariate model for ci + ct ≥ 2 at 6 months, in which stratification variables are included, identified treatment group—biopsy vs. control—(p = 0.03), donor age (p < 0.01), recipient gender (female) (p = 0.01), DGF (p = 0.03) and donor terminal creatinine (p = 0.02) as the only independent predictors of chronicity (Table 6). However, only 27% of the variance of the chronicity score was explained by these variables.

Table 6.  Chronic score at month 6—multivariate analysis (ITT biopsied)—using stepwise elimination with significance level as 0.05
Effectp-ValueOdd ratios95% Confidence interval
LowerUpper
  1. Note: Logistic model is used to assess the effect of baseline variables and donor's information on Ci + Ct score (<2 vs. ≥2) at Month 6. Stepwise selection method with significance level = 0.05 is used.

Treatment group (Biopsy vs. Control arm)0.03012.6231.097 6.272
Donor's age (> 50 vs. ≤ 50)0.00753.8771.43610.468
Recipient gender (Female vs. Male)0.00993.0971.311 7.313
DGF (Yes vs. No)0.02833.4911.14210.672
Donor terminal creatinine (> Median vs. ≤ Median)0.02440.4910.178 1.353
Donor terminal creatinine (Unknown vs. ≤ Median) 2.3060.824 6.450

The mean arteriolar hyalinosis score at 6 months was 0.36 ± 0.65 in the Control and 0.37 ± 0.65 in the Biopsy arm, which were unchanged from the scores observed in the implantation biopsies (0.42 ± 0.75 and 0.40 ± 0.69, respectively).

Renal function

The mean serum creatinine at 6 months was 119.90 ± 36.8 μmol/L in the Biopsy arm and 124.1 ± 41.2 μmol/L in the Control arm (ITT-biopsied group, p = 0.84). The calculated creatinine clearance at 6 months was 72.9 ± 21.7 mL/min in the Biopsy arm and 68.90 mL/min ± 18.35 mL/min in the Control arm (p = 0.18). The 24-h protein excretion was 0.21 ± 0.16 g/day in the Biopsy arm and 0.30 ± 0.37 g/day in the Control arm (p = 0.56). Similar results were obtained for creatinine clearance and 24-h protein excretion rates in the patients that had both baseline and 6-month biopsies (data not shown).

Safety considerations

Bleeding occurred in 5 of the 579 protocol biopsies for an incidence of 0.9%. All of these occurred in patients in the Control arm of the study. All bleeding episodes resolved within 3–22 days without the need of transfusion or patient hospitalization.

Other adverse events were not significantly different between the Biopsy and Control patients (data not shown). Specifically, polyoma virus nephropathy occurred in one patient in the Biopsy arm and in two patients in the Control arm.

Discussion

The principal finding of this randomized, multicenter study is that there is no benefit to the procurement of early protocol biopsies in renal transplant patients treated with TAC, MMF and prednisone, at least in the first 6 months of follow-up. The patients studied were at low immunological risk, as patients with high PRA, previous graft losses to early rejection and those at high risk for DGF (and consequently for rejection) were excluded from the study by design. The incidence of clinical rejections was low in both the Biopsy and Control arm patients, and the overall prevalence of subclinical rejection was only 4.6% in the Biopsy arm patients.

Since the initiation of this study, the prevalence of subclinical rejection in patients receiving TAC and MMF as baseline immunosuppression has been reported by several groups, mostly as uncontrolled studies, with or without antibody induction, and with biopsies obtained at various times posttransplantation (5–9). In aggregate, these studies indicate that subclinical rejection is less prevalent in patients treated with TAC and MMF-based regimens, as compared to those utilizing CsA and MMF, although there is some variability in the results reported.

In very early biopsies (performed at a mean time of 8 days posttransplant) in TAC plus MMF-treated patients Shapiro et al. reported borderline rejection in 21% of biopsies, and Banff type-I or type-II rejection in 25% (5). Similarly, Nankivell et al. reported subclinical rejection (including borderline) in >50% of patients at 1 month, with a virtual suppression of subclinical rejection at months 3, 6 and 12 (6). Gloor et al. reported subclinical rejection at 3 months to be 2.6% in patients who were the recipients of living-donor grafts (60%) and had received induction with anti-lymphocyte agents (>50%)(7). In studies that compared the prevalence of subclinical rejection between patients treated with TAC or CsA, Nankivell et al. reported subclinical rejection in 0.7% and 24% at 3 months in patients treated with TAC and MMF, and with CsA-ME and MMF, respectively (6). Similarly, Moreso et al. reported that in patients receiving TAC and MMF biopsied between 4 and 6 months posttransplantation the prevalence of subclinical rejection was 14.2%, as compared to 34.7% in patients receiving CsA-ME and MMF (8). Lastly, Rowshani et al. reported subclinical rejection in 15.2% of TAC and MMF and in 38.8% of CsA-ME and MMF-treated patients biopsied at 6 months (9).

In the present study the prevalence of subclinical rejection appeared to increase with time posttransplant, reaching up to 9% at 6 months. At this time-point, the mean TAC level was ∼9 ± 3 ng/mL and the mean prednisone dose was ∼0.1 ± 0.04 mg/kg daily. A similar increase in the prevalence of subclinical rejection following reduction of the steroid dose has been previously reported by Nankivell et al. in CsA-ME and MMF, although not in TAC and MMF-treated patients (6). Future studies that attempt calcineurin inhibitor (CNI) or steroid avoidance or withdrawal in renal transplant patients may require a protocol biopsy at appropriate time-points to ensure the safety of the immunosuppression regimen.

The primary endpoint of this study, the combined interstitial fibrosis/tubular atrophy score (ci + ct ≥ 2) at 6 months, was found in 34.8% of the Biopsy arm and 20.5% of the Control arm patients. The pathogenesis of early (i.e. before 6 months) interstitial fibrosis post-renal transplantation is multifactorial. By multivariate analysis, donor age, delayed graft function (or cold ischemic time), untreated subclinical rejection, clinical rejection (including vascular rejection), nephrocalcinosis and high levels of CNI have been implicated in some, but not all studies (10–14). Whether the early fibrosis observed in the present study is at least in part the result of CNI toxicity is not known. However, it should be noted that arteriolar hyalinosis, a potential marker of CNI, did not increase between the implantation and 6 month biopsies in either the Control or Biopsy arm patients.

In the present study, randomization to the Biopsy arm of the study, donor age, recipient gender (female), delayed graft function and donor terminal creatinine were shown to be the independent correlates of ci + ct ≥ 2. Although patients in the Biopsy arm had slightly more interstitial fibrosis than those in the Control arm, it is unlikely that this was due to the biopsies themselves. It is more likely that sampling error, the small increase in clinical and subclinical rejections sustained by the Biopsy arm patients, and perhaps other factors contributed to this unexpected result.

The prevalence of ci + ct ≥ 2 at 6 months observed in this study was greater than that observed in the earlier randomized study that utilized CsA and azathioprine as baseline immunosuppression (2). However in that study, the mean donor age was 10 years younger than in the present study and high doses of corticosteroids, which may suppress fibrogenesis associated with inflammation through their effect on NF-κB (15) were often given because of the high frequency of both clinical and subclinical rejection. Indeed, a small randomized study has suggested that a trend toward increased interstitial fibrosis occurred at 1 year in patients treated with TAC and MMF whose steroids were withdrawn after 7 days, as compared to a control group that remained on low-dose prednisone throughout the study, suggesting that steroid avoidance or withdrawal may be associated with increased interstitial fibrosis in such patients (16). An additional possibility to explain the decreased prevalence of fibrosis observed in the earlier randomized study may be that a calcium channel blocker (diltiazem) was given to all patients. Such agents may protect against tubular epithelial cell injury (17) and oppose CNI-induced vasoconstriction (18), and thus may have had beneficial effects on allograft histology. A recent randomized study has shown that use of the calcium channel blocker was associated with better renal function at 2 years in CsA-treated renal transplant patients, but renal biopsies were not performed (19). Whether calcium channel blockade would be protective in TAC-treated renal transplant recipients is not known, as renal blood flow appears not to be affected by TAC in both normal volunteers (20) and in renal transplant recipients (21) in contrast to CsA.

A potential limitation to this study is that the assessment of interstitial fibrosis was semi-quantitative, as it was done using the Banff schema. It is possible that more subtle differences in the fibrosis scores between the groups would have been detected with the use of a quantitative determination of fibrosis, as is possible with Sirius Red staining (22). However the extent of interstitial fibrosis determined by Banff criteria or Sirius Red staining correlates with both creatinine clearance and GFR (9,23), the former of which was similar in both patient groups.

Renal function at 6 months was very good, with both the Biopsy and Control arm patients exhibiting mean creatinine clearances of ∼70 mL/min, with negligible proteinuria. These results compare favorably with those of a recent multicenter study that compared TAC, MMF and prednisone versus TAC, MMF and daclizumab induction, in which the mean creatinine clearance at 6 months was reported to be ∼52 ±18 mL/min (24); but are similar to those reported by Rowshani et al., in which patients treated with TAC, MMF and prednisone had a mean serum creatinine clearance of 65 ± 17 mL/min at 1 year (9).

Despite the relative safety of the biopsy procedure (25, 26), that was confirmed in the present study, it is clear that both physicians and patients were reluctant to comply with the number of biopsies required of the patients randomized to the Biopsy arm of the study. It is our view, however, that protocol biopsies remain an indispensable tool in the monitoring of the renal transplant, the use of which may be restricted to patients with high immunological risk (e.g. patients that are sensitized to their donor), to patients in whom drug avoidance/minimization protocols are considered, and to patients with prolonged exposure to potentially nephrotoxic agents, as recently suggested by Racusen (27).

Acknowledgments

Funding for this study was provided by Astellas Pharma Canada, Inc. Data management and biostatistical services were directed by Miklos Schulz, PhD, of SciAn Services Inc., Toronto. The authors also thank Ms. Adrienne Groulx of SciAn for assistance with study design. The Data Safety Monitoring Board consisted of Pekka Haÿrÿ, MD, University of Helsinki, Helsinki, Finland (Chair) and Bryce Kiberd, MD, Queen Elizabeth II HSC, Halifax, Canada. The Writing Committee consisted of Drs. Catherine Girardin, Greg Knoll, Peter Nickerson and David Rush. The authors thank Ms. Evelyn Roloff for editorial assistance.

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